CA1094570A - Thiocyanato naphthalenes - Google Patents

Abstract

ABSTRACT
1-Thiocyanato-8-substituted naphthalene compounds and their use as biocides, especially for protecting aqueous media against infection by micro-organisms.

Description

Dx 29967 Thi~ invention relates to certain naphtbalene derivatives and their use as biocide6.
Many ~edia, and in particular aqueous media, are susceptible to attackjby micro-organi~m~. Such media include the cooling water systems of po~er station6, the water systems of paper mills, aqueou6 oil emulsions such a6 the cutting oils used as lubricants and coolants in the machininB of metals, water based paints and adhesives, and paint fil~g, Numerous compounds having diverse chemical Rtructures have been proposed for th~ elimination an~/or control of micro-organisms in a wnde ~ariety of different media, and these compounds are effective to a greater or lesser extent. The compounds of the present in~ention are very effective biocide~ against a wide spectrum of micro-organisms.

- 'lo'.~ ~S70 Dx 29967 According to the pre6ent invention there are provided 1-thiocyanato-8-substituted naphthalene compound6 of the formula:

R SCN
~ (I) wherein R is optionally substituted alkyl, optionally sub~tituted alkoxy, optionall~ substituted aryloxy, hydroxyl, halogen, nitro, optionally ~ubstitutedaryl, cyano, sulphonic acid, primary, secondary or tertiary amino, optionally substituted carbamoyl or optionally substituted sulph moyl, a~d wherein the ~aphthalene nucleus may carry other fiub6tituents and may ~orm part of a larg0r fused ring system.
The alkyl groups represented by R are preferably lower alkyl groupe, and ~ay carry 6ubstituents such as hydrox~, alkoxy or halogen.
Examples of 6uch alkyl and aubstituted alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-hydroxyethyl, 2-chloroethyl and

2-mothoxyethyl.
~he aryl sroups repre ented by R are preferably phenyl, which may be 6ubstituted by alkyl, alkoxy, ~itro or halogen. Exa~ples of ~uch substituted phenyl radicals are o-, m- and p-tolyl, o-, m- and p-methoxy-phenyl, o-, m- a~d p-chloro- (or bromo-) phenyl and o-, m- and p-nitrophenyl.
The alkoxy group~ represented by R are preferably lower alkoxy groups, for example, methoxy and ethoxy.

109 ~570 4 Dx 29907 The aryloxy groups repre6ented by R are preferably phenoxy sroups, which may be substituted by alkyl, alkoxy, nitro or halogen.
Examples of secondary and tertiary amino group6 represented by R
are methylamino, dimethylamino, ethylamino, diethylamino and phenylamino.
Th~ substituted carbamoyl or s~lphamoyl groups represented by R
are preferably of the formula -CONR1R2 or -S02NR1R2 wherein one of R1 and R2 is hydrogen and the other i8 lower alkyl, or R1 and R2 are each independently lower alkyl, or R and R2 together may form a five or six-membered heterocyclic rin6 including the nitrogen atom and which may include other hetero atoms. Examples of substituted car~amoyl or sulphamoyl groups are N-methylcarbamoyl, N,N-dimethylcarbamoyl, N-~ethyl-sulphamoyl, N,N-dimethylsulphamoyl, piperidinocarbonyl, piperidinosulphonyl, morpholinocarbonyl, morpholinosulphonyl, N-phenylcarbamoyl and N-phenyl-sulphamoyl.
Examples of the option~1 substituents which may be pregent on the naph-thalene nucleus are lower alkyl, lower alkoxy, hydroxy, halogen, nitro, optionally substituted amino, sulphonic acid, optionally substituted sulphamoyl andthiocyanato.
When halogen atoms are present in any of the compounds according to the pre~ent inYentiOn~ it is preferred that they are chlorine or bromine atoms.
The terms "lower alkyl" and "lower ~1koxy" in this specification mean alkyl and alkoxy groups respectively which contain from l to 4 carbon atoms.
The thiocyana~o group in the compounds of formula (I) may, in general, bc introduced b~ diazoti~atNi~on of a l-naphthylamine of the formula:
~

5 l 0 ~i~ S 7 0 ~x 29957 followed `~y reaction of the diazorium com~ound with an alXali metal thiocyanate, e.g. potassium thioc3ana'e, optionall~i together ~ith a transiti~ metal thiocyanate, e.g. cuprous thioc~anate. The amino group ~ay be introduced into the l-position of the naphthalene nucleus in a protected form to allo~
subse~uent introduction of the group R into the 8- position, the protecting group then being removed to liberate the free amino group w'nich is then replaced by the thiocyanato group as indicated previously. Alternatively, the group R may be present in the naphthalene nucleus before introduction of a protected amino group or other group which is ultimately replaced by the thiocyanato group.
Also compounds of the formula:
R SCN
~ X (II) wherein R has the meaning defined above, and X and Y each independently represent a hydrogen atom or a group as defined for R, and wherein R, X
and Y may be the same or different, provided that X and Y are not both hydrogen atom6, may be obtained by the reaction of a compound of the formula:

~ X (III) wherein R, X and Y have the meanings defined above, with thiocyanogen. The thiocyanogen m~y either be msde in situ, fsr example, by stirring the naphthalene compound of formula (III) with an inorganic thiocyanate and an inert solvent at a temperature below 20C and adding bromine dropwise to the mixture, or by addi~g a previously prepared solutior of thiocyanogen, for example, in carbo~ tetrachloride, to the naphthalene compound at a te~perature below 20C.

10~570 o v~ ,99~7 Other com~ounds o~ ~or~ula (I) lithin 'ne sco~e of the invention may be obtained by conventior~1 ~et~ods o~ orgaric che~nifitry.
~o- e~ample, halog~l ~toms may be introduced by dia7otisation of an appropriate amino derivalive followed by applica~lon of the Sardmeyer reaction. In this way 1-iodo-5-nitro-4-thiocyanatonaphthalene may be prepared from l-amino-5-nitro-4-thiocyanatonaphthalene. Elimination of the amino sroup from the latter compound via its diazonium derivative gives 1-nitro-8-thiocyanatonaphthalene, which may al80 be prepared by diazotisation of 1-amino-8-nitronaphthalene followed by reaction with an alkali metA1 thiocyanate. Similarly, l-amino-8-chloronaphthalene gives l-chloro-8-thiocyanatonaphthalene. Acylamino derivatives may be obtained by acylation of the corresponding amino compounds. Thus, reaction of l-amino-5-nitro-4-thiocyanatonaphth~lene with acetic anhydride in acetic acid gives the l-acetylamino derivati~e. l-Amino-5-nitro-4-thiocyanatonaphthalene may itself be obtained by direct thiocyanation of l-amino 5-nitronaphthalene; bis-thiocyanation gives l-amino-2,4-dithiocyanato-5-nitro~aphthalene, which may be converted into 2-amino-6-nitro-5-thiocyanatonaphtho~1,20d]thiazole by refluxing in an alcoholic solvent containing a ~m~ll quantity of a mineral acid.
LikewiRe, thiocyanation of l-amino-5-cyanonaphthalene give6 1-amino-5-cyano-4-thiocyanatonaphthalene; deamination of this compound via its diazonium derivative yields l-cyano-8-thiooyanatonaphthalene.

10~4570 7 Dk 29967 The compounds of formula (I) above are valuable biocides, and according to a further feature of the invention there i8 provided a method for protecting a medium which is susceptible to attack by micro-organisms against such attack, and controlling or preventing the proliferation of micro-organisms in a medium already infected thereby, which comprises adding to the said medium a biocidal amount of a l-thiocyanato-8-sub6tituted naphthalene compound as hereinbefore defined.
In the above definition, preventingthe proliferation of micro-organisms mean~ that the micro-organi6ms are not allowed to multiply further, the number of micro-organisms not necessarily being substantially altered. Gontrolling the proliferation of micro-organisms means that the rate of multiplication of the micro-organisMs is either reduced or rendered negative (i.e. a reduction in numbers, including the case of complete eradication), The biocides of the invention are particularly useful in controlling the growth of bacteria, fungi and algae in aqueous media, as hereinbefore defined,bt~t they also find application as paint film fungicides and algicides; in the pre~ention of fungal and/or bacterial attack on wood, hides and leather and for the in-can pre~erYation o~ water-based paints and adhesiveR.

10~4~70 8 Dx 29967 Preferred biocidal compounds within the scope of the preæent invention are l-thiocyanato-8-nitronaphthalene and l-acetylamino-5-nitro-4-thiocyanatonaphthalene, because of their greater effectiveness against a broad spectrum of micro-organisms.
The amount of biocide which is u~ed will depend upon the medium which is being treated and the nature of the micro-organisms involved, but for aqueous media an amount from l to 1000 parts per million by weight, based on the weight of the medium, q be used, from 25 to 500 parts per million by weight being generally effective, includi~g the case in which the aqueous medium i8 a water-based p~int.
When used a8 a paint film fungi~ide or algicide the biocide will generally be used i~ an amount to provide a concentratio~ in the paint, before it~ application to a substrate, f from 500 to lO,000 part per million by weight, based on the weight of the paint.
The biocidal compound of formula (I) may be addet to the appropriate ~edium as the undiluted æolid compound, as a solution in an organic ~olve~t, for example, a low~r alcohol 6uch as meth~nol or ethanol, or dimethylformamide, if desired together with water, or as a dispersion in water prepared with the aid o~ a dispersing ag~nt, for example, the sodium salt of a ~aphthalene-2Gulphonic acid/
for~aldehyde conden~ate.
Th~ method accordin~ to the present in~ention is particularly useful when the medium to be protected against infection by micro-organisms i8 a metal-wor~in~ nuid comprising a stabilised oil-in-water emulsion or a synthetic metal-working fluid, a paint film or water-based paint, and infection-susceptible media protected against infection by micro-organisms las4s70 g Dx 29967 by having incorporated th~rein a biocidal amount of a l-thiocyanato-8-substituted naphthalene compound as hereinbefore defined constitute a further feature of the in~ention.
The in~ention is illustrated but ~ot limited by the following Exa~pleR in which p~rts and percentages are by weight, and ppm means parts per million.
~Ea~
Pre~aration of l-amino-5-nitro-4-thiocyanatonaPhthalene 61.85 ~artY of lead thiocyanate and 395 parts of methanol were stirred at 10C and a 601ution of 25.53 parts of bromine in 158 parts of methanol was added to the 6uspension, maintaining the temperature at 5-12C. The thiocyanogen liquors 60 obtained were filtered from the lead salts. The thiooyanogen filtrate was kept at 5-15C.
25 Parts of l-amino-5-nitronaphthalene and 790 parts of metha~ol were stirred7 heated to re nux and filtered hot from a ~mall a~ount of impurity. The filtrate wa~ cooled to 10C.
The cold thiocyanogen solution was added to the stirred solution of ~-amino-5-nitronaphthalene, maintaining the temperature at ~-15C. The mixtuxe was stirred a further one hour at 5-15C and then made juæt aIkaline ~pH 7-8) by addit~on of concentrated a~monium hydroxide solution. The reaction mixture, after filtering from a small amount of insoluble material, was e~aporated to dryness~
The solid obtained was stirred with water, filtered, washed and dried.
The crude product was recry~tallised from toluene (usin2 charco~l~. The yield of fairly pure product was 17.8 g, m.pt 155~C.
An analytically pure sample was obtained by repeated recrystallisations from toluene and had m.pt 162C. The I.R. spectrum ~O~S70 10 Dx 29967 contained a band at 2150 cm 1 due to a thiocyanate group and bands at 3495 cm 1, ~39o cm 1 and 3240 cm 1 due to a primary aromatic amine ~roup.
Analysis, Found: C, 54.0; H, 3.1; N, 17.6; S, 13.6%
Cll ~ 302S requires C, 5~.9; H,2.9; N, 17.1;
S, 13.1~.
Exam~e 2 Pre~aration of l-nitro-8-thiocyanatonaPhthalene 24.5 Parts of l-amino-5-nitro-4-thiocyanato sphthalene (prepared as described above) were stirred with 524 parts glacial acctic acid at 650C and allowed to cool to room temperature. The suspension wa~ then added slowly to a well ~tirred solutio~ of nitrosyl sulphuric acid, (previously prepared from 13.8 parts sodium nitrite and 147 part~ of concentrated su~phuric acid) maintaining the temperature at 16-20C. After the completion of the addition, the diazo ~olution was allowed to stir for a further ~0 minutes.
28~6 Parts of cuprous oxide and 789 parts of ethanol were vigorausly stirred at 55C and the above diazo solution wa~
added dowly, mal~tainin~ the temperature at 55-600C. The reaction mixtur0 was then cooled, filtered and washed. The ~iltrates and washing~ were combined and drowned out in 10,000 part6 water.
Th~ suspe~sion was the~ filtered and washed well. The crude product was recryfitallised from ethanol (using charcoal) to yield 9.2 g product m.pt 118-119C. Th~ I.R. spectrum contained a thiocyanate band at 2160 cm Analy~is, ~ound: C, 57.9; H, 2.4; N, 12.2X.
ClIH6N202S requires C, 57-4; H, 2-6; N, 12-2~-~O~S70 11 Dx 29967 (N.B. a sample prepared from l-amino-8-nitronaphthale~e by diazotisation and reaction with inor~anic thiocyanates ga~e a product with the same physical properties as above.
Example 3 eparation of l_acet~lamino-7-nitro-4-thiocyanatonaRhthalene 24.5 parts of 1-amino-5-nitro-4-thiocyanatonaphthalene (pre F ed a~ described in ~xample 1) were stirred with 200 parts of acetic acid and 200 parts of acetic anhydride at room temp~rature for 16 hours. After drowning out the reaction mixture into water, the crude product was collected and recrystalli~ed from ethanol.
Yield 2g, m.pt a5-216C.
The I.R. spectrum contained a thiocyanate band at 2150cm 1 and bands at ~290cm 1 a~d 1660cm 1 due to an amide group.
An lysis, Found: C, 55.1; H, 3.1; N, 14.7X.
C13H~N30~S requires C, 54.4; ~, 3.1; N, 14.RX.

Preparation of l-iodo-~-nitro-4-thiocYanatonaPhthalene A ~olution of 2.45 parts of 1-amino-5-nitro-4-thiocyanato-` naphth~le~e in 50 F ts of glacial acetic acid wa8 diazotised with aoolution of nitrosyl~ulphuric acid, (previously prepared from 1.38 parts of ~odium nitrite a~d 16 F ts of concentrated ~ulphuric acid) maintaining the temFerature at 16-20~C. Tho diazo solution was then adde& to a solution of 3 F ts of potassi~m iodide in 20 parts of water and allowed to stir for a further hal~ hourO The crude product wa~
~iltered o~f, washed well, dried and recrystallised se~eral times from ethanol to yield 0.5g product, ~.pt 181-182C. The I.R. spectrum contained a thiocyanate band at 2160 cm 1.

: . :

0 9 4~ 0 12 Dx 2~967 Analysis, Found: C, ~7.1; H, 1.4; N, 7.7%
CllH5N202SI requires C, 37.1; H, 1.4; N, 7.9 Example 5 eparatio~ of l-c3ano-8-thioc~anatona~hth~lene Thi~ compound was prepared by thiocyanation of 4.18 part6 of l-a~ino-5-cyanonaphthalene, in a manner similar to that used to prepare l-amino-5-nitro-4-thiocy natonaphthalene in Example 1, to yield 3.4 F ts of crude l-amino-5-cyano-4-thiocyanatonaphthelene.

3.4 Parts of i~pure l-amino-5-cyano-4-thiocyanaton~phth~lene were then deaminated by the method described i~ Example 2 for deamination of l-amino-5-nitro-4-thiocyanatonaphthalene. Tho crude product, after drowning out into water, was filtered off, washed well, dried and recrystallised from ethanol to yield 0.62 parts of pure l-cyano-o-thiocyanatonaphthalene, m.pt 129.5-130.5C. The I.R.spectrum contained a band at 2220 cm 1 due to the cyano group and a band at 2160 cm 1 due to the thiocyanate group.
ADalySi8, Found; C, 68.o; ~, 2.7; N,13.0~
C12H6N2S requires C, 68~6; H, 2.9; 13-3%
Exam~le 6 Preparation of l-chloro-o-thio~yanatonaphthalene 3.92 parts 8-chloro-1-naphthylemine were stirred in dilute hydrochlorio at room temperature a~d diazotised by the careful addition of a ~olution of 1~52 partfi sodium nitrite in water. The diazo solution wa~ then added, at 10C, to a stirred su~pension of 3.5 parts potassium thiocyanate, 4.5 parts cuprous thiocyanate and 0.1 parts ferrous sulphate in water. After stirring the m xture for several hours, the product was filtered off and recrystallised several times from ethanol 10~570 13 Dx 29967 to yield 1 part of pure product, m.pt 77-80C. The I.R. spectrum contained a band at 2160 cm 1 due to the thiocyanate group.
Analysis, Found: C, 60.1; H, 2.8; N, 6.3; Cl, 16.1 C11~6ClNS requires C, 60.1; H, 2.7; N, 6.4; Cl, 16.2 xample 7 Pre~aration of l_amino-2,4-dithiocYanato-5-nitrona~hth~lene A solution of 9.4 p~rts of 1-amino-5-nitronaphthalene in 300 parts of glacial acetic acid was treated at 16-18C with a cold solution of thiocyanogen previously prepared by adding 16.8 parts of bromine in 70 parts of acetiG acid to 37.2 parts of lead thiocyanate in 160 parts of acetic acid.
The reaction mixture was poured into ice/water and the crude product was filtered off. After ~e~eral crystallisations from ethanol, the product was finally recrystpllised from aqueou~ acetone. Yield 3~ m.pt >250C. The I.R. spectrum contained band6 at 3460 cm 1 and 3360 cm 1 due to the amino group and bands at 2150 cm 1 and 2160 cm 1 due to the thiocyanate ~roups.
Analysis, Found; C, 48.7; H, 2.0; N, 18.2%.
c12H6N4o2s2 require6 C, 47.7; H, 2~0; N, 18.5 ExamPle 8 PreParatio of 2-~m no-6-nitro-5-thiocy~nato~1,2-d]thiazolc 0.25 Parts of l-amino-2,4-dithiocyanato-5-nitronaphthalene were heated in 25 parts of re$1uxing iRo-propanol oontaining 0.1 pa2ts of concentrated hydrochloric acid for ei8hteen hours. The ~uspan6ion wa~ drowned out into 250 parts of ioe/water. The essentially pure product was filtered off, washed and dried. Yield 0.22 parts, m.pt ~ 2850C. The I.R. spectrum contained bands at 3360cn 1, 3310 cm 1, 14 Dx 29967 3110 cm 1 and 1650 cm 1 due to a 2-aminothiazole ring, and a band at 2160 cm 1 due to one thiocyanate group.
Analyis, Found C,47.2; H, 1.9; N, 17.1; S, 20.9~
C12X6N402S2 require C, 47.7; H,2.0; N,18.5; S,21.2%
Example 9 In Vitro Bacteriostatic and F~tn~istatic Activit~_in A~ar l-Nitro-8-thiocyanatonaphthalene was dissolved in dimethyl formamide (DMF) (0.1 g/10 ml solYent) and added to nutrient agar to give a concentration of 100 ppm, and to malt agar to give concentrations of 100, 20 and 10 ppm.
The contr~l consisted of malt and nutrient agars containing 1 ml DMF
per 100 ml agar (i.e. the level of DMF in the 100 ppm biocide treatment).
The molten biocide-containing and control media werç poured into Petri dishe6 and allowed to solidify. Petri dishes containing each biocide concentration were poured in triplicate.
A Microtiter AM 80 Automatic Inoculator wa~ used for inoculating the p}ates. Nutrient agar plates were inoculated with the bacteria Pseudomonas aeruginosa, Escherichia coli, and Staph~lococcus aureus, and the malt agar plates were inoculated with the fungi, Ptllularia ~ullulans, As~er~illtls ~E~- Cladosporium 55~i3~ Alter~=ria tenuis, and Chaetomium ~lobosum.
The nutrient agar plates were incubated at 37C for 24 hours and malt a~ar plates were incubated at 25C for 48 hours, after which the plates were examined for the presence or absence of microbial growth.

~..

10~ 0 Dx 29967 Results .
Growth of Concentration Ps. E. S Asp. Clad Alt. Chaet.
of Biocide aerug coli aureus pullulans niger sphaer tenui~ globosum . .
100 ppm )_ _ _ _ _ _ 5 20 ppm )N OT T~S' 'ED _ _ _ _ 10 ppm )¦ l _ _ _ _ Control + ¦ + ¦ +

+, = Growth, i.e. organism not inhibited -, = no growth i.e. growth totally inhibited The~e result6 show that 1-nitro-8-thioc~anatonaphthalene has both antifungal and antibacterial activity.
Example 10 Determination of BactericidRl Activity of l-Nitro-8-thiocyanatonaphthalene Preparation of Reaction Mixture A 1~ solution of the biocide was prepared by adding 0.1 g 1-nitro-8-thiocyanatonaphthalene to 10 ml DMF. One-ml, 005 ml, 0.25 ml and 0.1 ml portions of the 1% solution were added to 100-ml volumes of sterile deionised water in 2SO-ml sterile cotton-wool plugged conical flasks to give final concentrations of 100, 50, 25 and 10 ppm of biocide. The control flask consisted of 100 ml water to which 1 ~1 of DMF had been added.
Preparation of Inoculum An 18-hour broth culture of Pseudomonas aeruRinosa was prepared by transferring one loop-full of a 24-hour broth culture irto 100 ml of nutriant broth in a 250-ml conical flask and incubating the latter in an orbital shaker at ~7~C. The 18-hour culture was diluted 1 in 10 in sterile deionised water and 1 ml of the latter suspension was used as the inoculum.

161 0 ~ ~ S 7 0 Dx 29967 Inoculation and Incubation o Reaction Mixture One ml of the inoculum was added to the 100-nl volumes of biccide solutions, and the latter were incubated statically at room temperature.
Det~rmination of Survivors Samples were removed from the biocide solutions after contact periods of 2, 4 and 6 hours, and after decimal dilution of the samples in physiological saline, s~rvivors were determined in nutrient agar (2 days at ~7C).
Re~ults Survivors (cells/ml) after contaot times of Concentration of Biocide _ _ 2 hour= 4 hours 6 hours 100 ppm ~10 C 10 ~ 10 50 ppm C10 < 10 < 10 25 ppm < 10 < 10 < 10 10 ppm2.0 x 101 < 10 < 10 ~c~l 2.9 x 106 3.6 x 106 7.1 x 106 These results show that l-nitro-8-thiocyanatonaphthalene is an active bactericidal agent.
~xamp~e 13 Determination of the Ability of l-Nitro-8-thiocyanatonaphth~lene to Withstand Sever~l Challenges of Bacteria Nutriert broth was prepared in sterile test-tubes containing 5, 10, 25, 50, 75 and 1~0 ppm of 1-nitro-8-thiocyanatonaphthalene~ The biocide was prepared in solution 20 times a~ strong as the above-mentioned concentrations, so that 1 ml of the biocide solutions added to 19 ml of nutrient broth gave the required final concentrations. The control consisted of nutrient broth with no biocide. ~ach concentration of biocide and the control was set up in triplicate~ ~ach nutrient broth tube was inoculated with 0~1 ml of a 24 hour broth culture o~ Pseudomonas aeruginosa and the inoculated broths were incubated at 37C for 24 hours, after which the tubes were examined for the presence or absence of growth before the second inoculation was made~
Altogether the tubes were inoculated four times.

10~ ~570 17 Dx 29967 Results Presence or Absence of Growth after Concentration -of Biocide 1st 2nd 3rd 4th Inoculation Inoculation Inoculation Inoculation . . .
lO0 ppm _ _ _ _ _ _ _ _ _ _ 75 ppm _ _ _ _ _ _ _ _ _ _ _ _ 50 ppm _ _ _ _ _ _ + + + + + +
25 ppm _ _ _ _ _ _ + + + + + +
10 ppm _ _ _ + + + + + i + + +
5 pPm + + + + + + + + + + + +
lO Control ~ + + + + + + + + + + +
(no biocide) , ~ . . . ~
The result~ show that the higher levels of l-nitro-8-thiocyanatonaphthalene (e.g. the lO0 ppm level) control at least four bacterial challenges. The chemical i6 therefore a stable antibacterial agent with high activity.
15Example 12 Com~arison of the In-Vitro Fun~icidal Activities of l-Nitro-8-thiocyafiatonaphthalene eParation of Reaction Mixture A 1~ solution was prepared by dissolving O.l g of the solid compound in lO ml DMF. 0.5-ml and l.0-ml volumes of the above-mentioned 1~ solutions were added to lO0 ml volumes of sterile deionised water in sterile cotton wool plugged 250-ml conical flasks to give solutions of 50 and lO0 ppm biocide. The control flask consisted of lO0 ml water.

10~9~S70 18 Dx 29967 Preparation of Inoculum 1 cm-diameter discs were cut from cloth and transferred into water in a conical flask, and sterilised by autoclaving for 15 minutes, at 15 p.s.i. The discs were then transferred onto malt agar in Petri dishes (6 per Petri dish) and inoculated with a spore suspension of Asper~illus ni~er by spraying the plate and contents from an atomi~r. The inoculated discs were incubated for 24 hours at 25C, after which time they were overgrown with mycelium but there were no spores pre~ent, Inoculation and Incubation of Reaction Mixture All reaction mixtures were maintained at 30C in an orbital ~haker. At zero time six fungus-impregnated discs were transferred into each flask.
Determination of SurYivor~
After contact periods of 3, 6 and 24 hours two of the discs were remo~ed from each flask and transferred into 100 ml of sterile water in a sterile bottle. The latter was momentaily shaken and after a 30-minute periocl the two discs were remo~ed from the water and tran6ferred on to malt agar in a Petri dishO All Petri dishes containing the fungu6-impregnated di~cs which had been subjected to biocide treatment were incubated for 3 days at 25C. The plates were examined for the presence or absence of fungal growth. The presence of Pungal growth indicates that the biocide treatment had not totally killed the mycelium on the discs, whereas the absence of growth indicated total kill by the biocide treatment.

109~570 19 Dx 29967 Results .
Survival of fungi in biocide solutions after Concentration of Bioci~e . . 3 hours 6 hc urs 24 h ourR
Repl.1 Repl~2 Repl.1 Repl.2 Repl.1 Repl.2 100 ppm 3 3 1 0 0 0 50 ppm 3 3 3 3 0 0 Control 3 3 3 3 3 - In the above table:-~; fungal Browth over entire disc and readily spreading over surrounding agar (i.e. no kill).
2; fungal growth esqentially confined to the disc.
l; fungal growth over up to h~lf of the area of the disc only (partial kill) O; no fungal growth on disc (i.e. total kill).
The results show that l-nitro-8-thiocyanatonaphthalene has fungicidal activity.
Example 13 Activity of l-Nitro-8-thiocyanatonaphthalene in ControllinR Bacterial Growth in Metal-Workin~ Fluids The biooide, in the form of a 1~ solution in DMF, was addçd to 100 ml volumes of a 1 in 20 oil in water emulsion prepared from Progol 44 emulsifiable oil (Mobil) to give final concentrations of 50, 100 and 200 ppm in the emulsion.
The biocide-containiDg emulsions and bioSide-free controls in 250 ml cotton-wool plugged conical flasks were incubated at 30C
on a rotary ~haker. The emulsion~ were inoculated once weekly with 0.5 ~1 of an overnight broth culture of Pesudomonas aeru~inoRa and sur~ivors were lO~ ~S70 Dx 29967 determined after 1 and 3 days in each wee~. Survi~ors were determined in nutrient agar by dec mal dilution of 1 ml samples, removed from t~e emulsions.
Results Survi~ors (cells/ml) in Concentration Week 1 Wee~ 2 of Biocide Day 1 Day 3 Day 1 Day 3 . . . - - . , .
50 ppm ~ 10 1.2 x 102 9.6 x 104 > 3.0 x 105 100 ppm ~ 10 < 10 ~ 10 < 10 200 ppm ~ 10 < 10 ~ 10 < 10 Control 7.1 x 106 6.2 x 107 6.1 x 107 6.7 s 107 The results indicate that l-nitro-8-thiocyaDatonaphthalene has high antibacterial acti~ity in an oil-in-water emNlsion.
ample 14 Activity of l-nitro-8-thiocyanatonaphthalene as a Paint Film FungLcide.Tncor~oration of Bioc_de into Paint A 0.09 g quantity of the biocide was added to 18.9 g mill base, and mixed thoroughly by using a high speed micro shear stirrer.
11.1 g of PVA copolymer emulsion was then ~lowly stirred into the biocide-containing mill baæe by hand. The biocide was therefore present in the fiDal emulsion paint at a concentration of3000 ppm. The paint sample was then stored in a tightly~sealed plastic container at 50C for 2 weeks~ The control consisted of paint containing no biocide.
A~plication of Biocide-Containing Paint on Filter PaPer and Subseauent Treatment Two coats of paint were applied by brush to one side of two 7-cm diameter Whatman No. 1 filter papers, Allowing 24 hours for drying between coats. The filter papers were placed in an oven at 50C for 24 hours and then 10~70 21 Dx 29967 transferred to a leaching apparatus for 24 hours in which a fine mist of water was sprayed over the filter papers.
The weathered filter papers were then ethylene oxide sterilised~ after which they were transferred on to malt agar in a Petri dish. For ~ch paint variant one filter paper was inoculated with a spore 6uspension of Alternaria tenuis and the second filtrr paper was inoculated with a ~le~De~E~ sphaerospermum spore suspension. The inoculated Petri dishes were then incubated for 5 days at 25C, after which the filter papers were examined for the presence or ab~ence of growth.
Results .. . .
Growth on Filter P~per of:-Alternaria Cladosporium tenuis 6~haerospermum Biocide O O
Control _ __ ~ _ __ _ _ 2 3 , Growth over the entire surface of the filter paper 2 = Growth over 50~ of the filter paper O = No growth; i.e. total inhibition of fungal growth by the biocide.

The results show that l-nitro-o-thiocyanatonaphthalene has high activity as a paint film fungicide.

109 ~S70 22 Dx Z9967 Example l~
The procedure described in Example 9 was repeated, co~par~ng the following thiocyanatonaphthalenes for antibacterial and ~ntifungal activity:
A. l-Nitro-8-thiocyanatonaphthalene B. l-Acatylamino-5-nitro-4-thiocyanatonapthalene C~ l-chloro-8-thiocyanatonaphthplene D. l-Ami~o-5-nitro-4-thiocyanatonaphthalene B~ l-Iodo-5-nitro-4-thiocyanatonaphthalene F. l-Amino-2,4-bis(thiocyaDato)-5-nitronaphthalene G. 2-Amino-6-nitro-5-thiocyanatonaphtho[l,2-d~thiazole H. l-Cyano-o-thiocyanatonaphthalene The following results were obtained:

trat;on Growth of (ppm) r-~ __ . ~. . ~ _ Ps. E. S. P~ Asp. Clad. Alt. Chaet.
aerug coli aureus luanl8u_ niger sphaer tenuis globo6um ~ A ~ ¦ ~ - r C 100 + + ~ _ + + ~ +
D lO0 + + + _ + _ _ +
~ lO0 _ _ + _ _ _ _ F lO0 _ ~ + _ _ + _ _ G 200 ~ , ¦ +
Control ~ + + + + + +
(no biocde) _ . ~ . ~ . .

iO~4~70 23 Dx 29967 In the above table - and + mean the absence or presence, respectively, of bacterial or fungal growth.
From these results it can be ~een that compounds A, B and E have a broad ~pectrum of activity against both baetoria and Pungi, whilst the remaining compounds ha~ anti-fungal rather than antibacterial acti~ity, although F i8 acti~e against Ps.aeruginosa.
Example 16 Bacter~eidal Acti~ity of l-Nitro-8-thiocyanatonaPhthalene (A) and l~Aeetylamino-5-nitro-4-thioeyanatonaPhthalene (B) in a Coolin~ Tower Water An artifieial cooling tower water was prepared from:
Solution 1 : Calcium ehloride 10.769 g Mag~esium chloride 6.782 g Water 100 ml andSolution 2 : Sodium bicarbonate 5.60~ g Water 100 ml by mixing 4 ml of Solution 1, 8 ml of Solution ~ and 2 ~1 of ethylene glyool ancl maXing the mixture up to 2 litres with distilled water.
The solution was trans~erred in 100 ml portion~ into bottles, and autoelaved to sterilise the bottles and contents.
The pH of the solution was 8.5.
The test ehemic21s A and B9 as 1~ solutions in dimethyl-for~amide, were added to 100 ml volume6 of the artifieial cooling tower water in 250 ml sterile conical flasks plugged with eotton wool, to give coneentration of aoti~ ingredient of 50 and 100 ppm. ~he 10~94570 24 Dx 29967 control consisted cf cooling tower water with no biocide.
The fla6ks were transferred to an orbital shaker at 30C and, after a 30 minute equilibration period, were inoculated with 1 ml of an 18 hour broth culture of Pseudomonas 5aeruRinosa.
After incubation periods of 1, 2 and 4 hours one-~l volumes ~ere removed from each flask, decim~lly diluted in physiological ~alin~, and surviving bacteria were determined on nutrient aear. After incubation of the nutrient a~ar plates for 2 days at 37C, the survi~ing bacteria were enum~rated.
Reeults were as follows:
r . _ Treatment Survi~ors (cells/ml) after:
. . ,- . .
1 hour 2 hour~ 4 hours " . .~_.
Compound A, 200 ppm c 10 c 10 ~ 10 15 100 ppm c 10 c 10 c 10 50 ppm c 10 c 10 c 10 Compot~d B~ 200 ppm 2~0 x 104 c 10 c 10 100 ppm 6.o x 104 c 10 c 10 5 ppm 5.0 x 104 c 10 c 10 20Control 3.0 x 103 2.5 x 108 These refiults show that Compounds A and B have high activity as cooling tower water biocides.

10~570 25 Dx 29967 Example 17 Bactericidal Acti~ity of l-Nitro-8-thiocyanato-naphthalene (A) and l-Acetylamino-5-nitro-4-thiocyanato-naphthalene (B) in Paper Mill Water An artificial paper mill "white water" was prepared having the following composition:
Starch -35 g Clay -35 g Titanium dioxide 0.023 ~
Animal ~lue 0.001 g Ky~ine resin 0.34 g Sodium aluminate 0.023 g Rosin size 0.023 g Glucose 2.84 g Peptone 1.5 8 10~ ~round wood pulp~ 37 ~1 Water to 1 litre 10 g dry wood in 100 g suspension This medium wa~ dispensed in lC0 ml volumesl autoclaved at 1.05 kg/cm2 for 15 minutes and transferred into 250 ml ~terile conical flask~.
The te6t chemicals A and B as 1% solution6 in dimethyl-formamide were added to 100 ml volumes of white water medium to give concentrations o~ active i~gredient of 50 and lC0 ppm, 1 ml of an 18 hour culture of ~terob~cter cloacae was added, and the inoculated media were incubated in 250 ml flasks in a shaking water bath at 30C.
The control consi~ted of the white water medium without biocide.

1(~9~570 26 Dx 29967 After incubation periods of 1,2 and 4 hours, one-ml volumes were re00ved from each flask, decimally diluted in phy~iological saline, and surviving bacteria were determined on nutrient agar. After inGubatio~ of the nutrient agar plate6 for 2 days at ~7C the surviving bacteria were enumerated.
Resultæ wçre as follows:

Treatment - )r~ ~0~114/ml) afte r 1 hour 2 hours 4 hours Compound A, 50 ppm ~ 10 ~ 10 c 10 100 ppm 1.2 x 1026.6 x 102 c 10 Compound B, 50 ppm 4.2 x 103 2.3 x 102 8.1 x 10' 100 ppm 5.~ x 1034~0 x 10~ 3.9 x 10' Control r 4~1 ~ L07 1 5.6 x 1078.2 x 107 These results show that Compounds A and B have both markedly reduced the bacteriel count a~d are effecti~e biocides in paper mill water.
Example 18 Antibacterial Actiqity of l-Nitro-8-thiocyanatonaphthalene (A) i~ Oil Rec ver Water An artificial oil recovery water (see water medium) having the following composition va~ prepared:
Dipota~sium hydrogen pho~ph~te 0.5 g Ammonium chloride 1.0 g Sodium sulphate 1.0 g Sodium lactate (70~) 5.0 ml Yeast extract 1.0 g Sodium ascorbate 0.1 e Sodium thioglycollate 0.1 g 109~570 27 Dx 29967 Ferrous sulphate 0.5 g Aged Rea water ~ to 1 litre 75~ sea water; 25% distilled water. Ihe sea water was previou61y aged by storage in the dark for 1 month at room temperature.
Thi8 medium waa dispensed in 18 ml volume5into screw capped bottles and autocla~ed to sterilise the bottles and oo~tents.
After the a~toclave treatment 1 ml of an aqueous solution o~ 60dlum sulphite was added to each bottle to giYe a final concentration of 10 pp~.
Compound A and sterile distilled water were then added to give a fi~al volume of 20 ml in each case, and concentrations of 5, 10 and 50 ppm of Compound A. The control consi~ted of the sea water ~edium and 60diu~ 6ulphite, without biocide.
Each bottle containing 20 ml ~dium wa8 inoc~lated with 0.2 ml Desul~ho~ibrio ~. and inoubated for 5 days at 25C, after which the media were examined for the presence or absence of bacterial growth. Each test was carried out in triplicate.
Re~ults were aa follows:
. ~ ~ ~
Treatment Growth of Desulphovibrio Compound A 50 ppm _ _ _ 10 pp~ _ _ _ 5 ppm + + +
Co~trol In the above table - and + mean the ab6ence and presence of bacterial growth, re6pectively.
The~e re~ults show that at a concentration of 10 ppm lO~ ~S70 28 Dx 29967 Compound A controls the growth of Desulphovibrio and ha6 utility as a biocide in oil recovery water.
Example 19 Antibacterial Activity of l-Acetylamino-5-nitro-4-thioc~a~natona~hthalene (B) a6 an In-C~n Paint Pre6ervative Compound B, a6 a 1~ solution in dimethylformamide, was added to 50 g quantities of a styrene-acrylic emulsion paint to give final concentration6 of lO0 and 200 ppm. The control consisted of paint co~taininæ 1 ml of dimethylformamide (th~ amount required to provide the 200 ppm le~el of biocide concentration) but no biocide.
The paint samples were each inoculated with 0.5 ml of a mixture of overnight broth cultures of Ps~ ~E9e~ae~ E-coli and Enterobacter cloacae once weekly for 2 weeks. Survivin~ bacteria were determined each week, l and 3 day6 after inoculation, in nutrient agar by the decimal dilution method.
Re~ults wer~ as follows:-., , . ~ . .
Treatment Survivors (cells/~ paint) in - ~ Z D~ 3 Compound B 200 ppm c lO c 10 ¢ lO c lO 4 lO0 ppm 5.6 x 105 l.0 x 105 7.0 x lO ~.0 x lO
_ _ _ _ _ _ _ _ 5.9 x lO6 2.0 x 107 2.5 x 107 4.0 x 106 The~e results show that at a level of 2Q0 ppm Compound B
has hi~h activity as an in-can paint pre6ervative.

10~4S70 29 Dx 29967 Example 20 Activity of 1-Acetylamino-5-nitro~4-thiocyanatona~hthalene (B) as a Metal Workin~ Fluid Biocide Compound B, as a 1~ solution in dimethylformamide, was added to 100 ~1 ~olume6 of a 5% oil-in-water emulsion prepared from Prosol 44 (Mobil) e~ulsifiable oil, to giYe f~n~l concentrations of 100 And 200 ppm of active ingredient. The control consisted of 2 ml of dimethylformamide in 100 ml of the emulfiion (i.e. the amount of dimethylfor~amide added to the emul~ion to give the 200 ppm concentration of biocide).
The 100 ml volumes of emulsion in 250 ~1 conic~l flasks plugsed with cotton wool were inoculated with 0.5 ml of an 18 hour broth culture of Pseudo~onas aeru~inosa,transferred to an orbital shaker and incubated at 30C. The test emulsions were inoculated once weekly for 3 weeks. Surviving bacteria were determined on nutrient agar by the deci~al dilution procedure after incubation periods of 1 and 3 days in eac}
week.
Results were as follow6:
, .
Sur~iY or6 (cell~/ml) af ter Treatment Week 1 Week 2 Week 3 Day 1 Da~ ~ Da~ 1 Da ~ Da~ 1 Day 3 C~mpound B 100 ppm c 10 ~ 10 c 10 ~ 10 3.9x104 c 10 200 ppm ~ 10 c 10 ~ 10 c 10 c10 c 10 r~l 1.64x10~ 1.54x107 8.9xlo7 1.3~xlo8 ~2 ~9C8 2.8x10 Thesc re~ult6 show that Compound B is highly active as a metPl working fluid biocido.

10!~4S70 30 Dx 29967 Example 21 Al~icidal Activit~ of l-Nitro-8-thioc~anatonaphthalene (A) and l-Acetyla~ino-5-nitro-4-thiocyanatonavhthalene (B) Ank~strodes~u6 spirilliformis, Stichococcus bacillaris and Ghlamydomonas reinhardii were inoculated into one-litre batches of Difco algal broth and incubated at 15C for 2 weeks in an illu~inated refrigerator. The three cultures were then combined and the mixed culture was disponsed in 50 ml volumes into 100 ml sterile conical flasks.
Compounds A and B were added to gi~e final concentrations of 0~1, 0.5, 5.0 and 10.0 ppm.
The test samples were incubated for up to 2 weeks in the illuminated re~rigerator at 15C, and the nasks were examined for the presence or absence of a green colouration. The retention of the green colour indicated tb~t the algae remained ~iable, whereas lack of colour of the sample indicated that the algae had been killed.
Reaults were a~ ~ollow6:-Minimum Lethan Concentration ~ (ppm) after Treatment _ 3 Days 7 Days 14 Days _ _ Compound A 1.0 0.1 0.1 2 0 a ~ ~ 3 I ~ 10 r . 10 ~ i.e. the minimum concentration of biocide at which no green colouration was obser~ed.
These realts sho~ that Compou~ds A and B both ha~e antialgal actl~ity.

Claims (13)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. 1-Thiocyanato-8-substituted naphthalene compounds of the formula:
where R is alkyl, alkoxy, aryl, aryloxy, hydroxy, halogen, nitro, cyano, sulphonic acid, primary, secondary or tertiary amino, carbamoyl or sulphamoyl and wherein the naphthalene nucleus may carry other substituents selected from the group consisting of lower alkyl, lower alkoxy, hydroxy, halogen, nitro, amino, acylamino, sulphonic acid, sulphamoyl and thiocyanato.

2. A naphthalene compound according to Claim 1 wherein R is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-hydroxyethyl, 2-chloro-ethyl, 2-methoxyethyl, methoxy, ethoxy, phenyl and phenoxy groups.

3. A biocidal 1-thiocyanato-8-substituted naph-thalene compound wherein the substitution in the 8-position is selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, hydroxy, halogen, nitro, cyano, sulphonic acid, primary, secondary or tertiary amino, carbamoyl and sulpha-moyl.

4. A biocidal 1-thiocyanato-8-substituted-naphtha-lene compound according to Claim 3 wherein alkyl is lower alkyl or substituted lower alkyl the substitution being selected from the group consisting of hydroxy, alkoxy and halogen, aryl is phenyl or substituted phenyl, the substitution being selected from alkyl, alkoxy, nitro and halogen, aryloxy is phenoxy or substituted phenoxy the substitution being selected from the group consisting of alkyl, alkoxy, nitro and halogen and the sulphamoyl and carbamoyl groups having the general formulae -SO2NR1R2 and -CONR1R2 respectively wherein and R2 are selected from the group consisting of hydrogen and hydrogen, hydrogen and lower alkyl, lower alkyl and lower alkyl and hydrogen and phenyl, or R1 and R2 together are selected from the group consisting of -(CH2)5- and -(CH2)2-O-(CH2)2-.

5. A biocide according to Claim 3 which is further substituted in the naphthalene ring in other than the 1- and 8-positions by at least one member of the group consisting of lower alkyl, lower alkoxy, hydroxy, halogen, nitro, amino, acylamine, sulphonic acid, sulphamoyl and thiocyanato.

6. 1-Thiocyanato-8-nitronaphthalene

7. 1-Acetyl-5-nitro-4-thiocyanatonaphthalene.

8. A process for the manufacture of the 1-thio-cyanato-8-substituted naphthalene compounds claimed in Claim 1 and having the formula:

wherein R has the meaning stated in Claim 1 and X and Y each independently represent a hydrogen atom or a group as defined for R, and wherein R, X and Y may be the same or different, provided that X and Y are not both hydrogen atoms, which com-prises reacting a compound of the formula:

wherein R, X and Y have the meanings stated above, with thiocyanogen.

9. A method for protecting a medium which is sus-ceptible to attack by micro-organisms against such attack, and controlling or preventing the proliferation of micro-organisms in a medium already infected thereby, which com-prises adding to the medium a biocidal amount of a l-thio-cyanato-8-substituted naphthalene compound as claimed in Claim 1.

10. A method as claimed in Claim 9 wherein the medium is an aqueous medium, the thiocyanato-naphthalene compound being added in an amount from 1 to 1000 parts per million by weight based on the weight of the medium

11. A method as claimed in Claim 10 wherein the thiocyanatonaphthalene compound is added in an amount from 25 to 500 parts per million by weight, based on the weight of the medium.

12. A method as claimed in Claim 10 or 11 wherein the aqueous medium is a water-based paint, a metal-working fluid comprising a stabilized oil-in-water emulsion or a synthetic metal-working fluid.

13. A method as claimed in Claim 9 wherein the medium is a paint film, tha thiocyanatonaphthalene compound being used in an amount to provide a concentration in the paint, before its application to a substrate, of from 500 to 10,000 parts per million by weight, based on the weight of the paint.